The present invention relates to analyzing, monitoring and controlling chemical reactions and, more particularly, but not exclusively to systems and method for identifying specific points in chemical reactions, such as a point in a chemical reaction, in which a new stage of the chemical reaction begins.
Chemical reaction may need to be characterized in real-time.
For example, detection and quantification of a molecule in a chemical reaction may be required to take place as the reaction progresses, in order to characterise the pattern of the reaction, take certain steps when the reaction moves into a new phase, etc.
Of special interest are a point of start of exponential growth of the chemical reaction product and a point where growth of the product begins to slow to a halt, also referred to as elbow points. The elbow points may be used to determine whether any reaction products have been produced. The magnitude of the reaction may be determined using a measured physical property of the reaction, say photometric measurements between the elbow points, as described in further detail hereinbelow.
One widely used and well-established laboratory technique is Polymerase Chain Reaction (PCR).
In PCR, the polymerase enzyme attaches to a target DNA sequence and replicates it exactly along with its containing chromosome or DNA strand. If the target DNA sequence is not present or for some reason is unavailable for attachment to the PCR enzyme, no replication of DNA takes place. This procedure is repeated many times in a PCR reaction instrument. Thus, the target DNA sequence, as well as overall DNA concentration, is amplified to microgram levels to allow for accurate detection and data analysis.
Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) is a widely used PCR method. QF-PCR is commonly used for diagnosis and research in fields such as disease (infectious or inherited), blood screenings, histology, oncology, tissue typing and drug discovery.
In QF-PCR, phosphate groups are introduced into the PCR reaction in order to mark the replicated molecules for purposes of real-time detection and quantification.
The two common methods for QF-PCR are: 1) Fluorescent dyes that intercalate with double-stranded DNA and 2) Modified DNA probes that fluoresce only when hybridised with the target DNA sequence, as known in the art.
The latter method is more sensitive and therefore more reliable and accurate, it also allows for real-time quantification of multiple DNA sequences using differently coloured probes.
The data received is in the form of fluorescent intensity, called FI.
The FI data may be represented using a graph. The shape of the graph may be either linear (if a target DNA sequence was not found or not amplified) or appear to be a sigmoid curve (if the target sequence DNA was amplified).
In case of presence of the target DNA sequence, there arises a need to identify the point where amplification of the DNA sequence begins to take place, also referred to as the threshold point or CT. However, the FI data in the threshold's region usually has a low Signal to Noise Ratio (SNR). Consequently, determining CT with a high degree of accuracy requires a method or a combination of methods for refining the FI data.
In photometric methods such as QF-PCR, photometry is utilised for real-time detection and quantification of a reaction product.
In order to determine whether a) any reaction products have been produced, and b) the magnitude of production, targeted photoactive probes are utilised in the chemical reaction, to produce a photometric effect (i.e. light) detectable by an optical sensor. The magnitude of the production is derived from data pertaining to the intensity of the photometric effect.
Determining whether any reaction products have been produced, and the magnitude the production accurately is limited by the amount of noise in the recorded photometric data. The noise may originate from chemical sources, such as the reaction mix, as well as from electronic sources, such as the instrument used for light detection.
Several traditional methods have been used to determine time points of exponential growth on a graph representing a quantitative measurement of a chemical reaction over time.
One traditional method involves an n-derivative of light intensity used to determine time periods of exponential growth.
International Patent Application No.: PCT/US2002/031144, to Taylor et al., published on Apr. 10, 2003, entitled “Adaptive baseline algorithm for quantitative”, describes baseline subtraction algorithms developed to reduce tube-to-tube and cycle-to-cycle variabilities during real time PCR amplification. Particularly, Taylor describes an algorithm for determining a threshold cycle, for detection of an amplified nucleic acid production.
U.S. patent application Ser. No. 11/645,964, to Woo et al., filed on Dec. 27, 2006, entitled “Automatic threshold setting and baseline determination for real-time PCR”, discloses a method which involves a base-lining operation, for identifying the bounds of a baseline region and performing a linear interpolation to identify the characteristic equation defining the baseline.
U.S. patent application Ser. No. 11/316,315, to Kurnik et al., filed on Dec. 20, 2005, describes Systems and methods for determining characteristic transition values such as elbow values in sigmoid or growth-type curves, utilizing a Levenberg-Marquardt (LM) regression process.
U.S. patent application Ser. No. 11/861,188, to Kurnik et al., filed on Sep. 25, 2007, entitled “PCR elbow determination using curvature analysis of a double sigmoid”, describes a method utilizing a first or second degree polynomial curve that fits the a growth type curve, and determination of a statistical significance value for the curve fit. The significance value indicates whether the data represents significant or valid growth.
Some traditional methods based on linear regression, are used to determine the time point where growth in light intensity changes from linear to exponential. Typically, the linear regression based methods include prior setting of a threshold for intensity, to determine the start of exponential growth.
A particular method involving two-phase regression is described in an article by Edna Schechtman, published in the Journal of Statistical Computation and Simulation, volume 17, issue 3, 1983 (pages 223-229), entitled “Inference in Two-Phase Regression: A Simulation study with Non-normal Observation”.
Some currently used methods involve converting data into a graph image and rotating the image.
In a one example, U.S. patent application Ser. No. 11/349,538, to Kurnik, filed on Feb. 6, 2006, entitled “PCR elbow determination by rotational transform after zero slope alignment”, describes PCR data set visualization in a two-dimensional plot of fluorescence intensity vs. cycle number. Then, the PCR data set is adjusted to have a zero slope.
In a second example, Japanese Patent Publication No. 2007128483, to Kurnik, published on May 24, 2007, entitled “PCR elbow determination by rotational transform”, describes a rotation transform application to a modified data set, to rotate the data about a defined coordinate such as the origin, so that the data point representing the Ct value may become a minimum or a maximum along the intensity axis. The data point representing the elbow or Ct value of the curve is identified, and this data point is then reversed back and the cycle number of the data point is displayed.
U.S. patent application Ser. No. 11/423,377, to Kurnik, filed on Jun. 9, 2006, entitled “CT determination by cluster analysis with variable cluster endpoint”, describes PCR data set visualized in a two-dimensional plot of fluorescence intensity (y-axis) vs. cycle number (x-axis). Then, the points of the plot are clustered. Using the identified clusters, a linear slope of each of the clusters is determined and the data point representing the elbow or Ct value of the PCR curve is identified as an end point of one of the identified clusters.
Other Methods, such as the one disclosed by Wittwer et al., in U.S. Pat. No. 6,503,720, combine two or more of the methods described hereinabove.